EP2718424B1 - Formulation et procédé de passage et de collecte de cellules souches pluripotentes humaines - Google Patents

Formulation et procédé de passage et de collecte de cellules souches pluripotentes humaines Download PDF

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EP2718424B1
EP2718424B1 EP12723066.2A EP12723066A EP2718424B1 EP 2718424 B1 EP2718424 B1 EP 2718424B1 EP 12723066 A EP12723066 A EP 12723066A EP 2718424 B1 EP2718424 B1 EP 2718424B1
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formulation
sodium citrate
cells
stem cells
culture
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EP2718424A1 (fr
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Ying Nie
Jonathan Allen Rowley
Thomas Fellner
Patrick Walsh
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Lonza Walkersville Inc
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0607—Non-embryonic pluripotent stem cells, e.g. MASC
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin

Definitions

  • the present invention relates to a formulation and method for harvesting/passaging pluripotent stem cells. Specifically, the present invention relates to a method using formulations including sodium citrate.
  • hPSCs Human pluripotent stem cells
  • hESCs human embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • Scaling up existing flask-based processes is a critical stepping stone in translating current hPSC research into clinical application.
  • One of the biggest challenges is to establish a scalable passaging method for large scale multilayer vessels that maintains high yield, pluripotent phenotype, and karyotypic stability.
  • hPSCs are harvested and passaged as colony fragments by mechanical scraping with or without pre-treatment with enzymes (such as collagenase or Dispase®).
  • enzymes such as collagenase or Dispase®
  • This process is labor intensive and cannot be applied in culturing hPSCs in multilayer cell culture vessels, the platform widely used in producing commercial scale adherent cells. Cells growing in multilayer cell culturing vessels cannot be accessed for scraping.
  • mechanical scraping causes damage to cells. Without scraping, cell viability can increase up to 90 percent.
  • Known methods related to single-cell passaging and harvesting are not desirable due for example, to concerns related to low post-passaging and cryopreservation recovery and abnormal karyotype, associated with low cloning efficiency of hPSCs.
  • Rho-associated kinase inhibitors have been reported to be able to improve hPSC cloning efficiency, the mechanism is not fully understood and the effect of ROCK Inhibitors on hPSC culture is yet to be evaluated. Therefore, passaging hPSCs as single cells in the presence of ROCK Inhibitors is not widely accepted.
  • EDTA ethylene diamine tetraacetic acid
  • the typical procedure of passaging hESCs with Versene® EDTA starts with washing the culture with Ca2+/Mg2+-free buffer (for example, Dulbecco's phosphate -buffered saline; DPBS), followed by incubating the culture in Versene® EDTA for 4-9 minutes. Versene® EDTA is then removed and cells are physically removed from the surface as clusters by manual hosing of the cells with culture medium via pipetting.
  • Ca2+/Mg2+-free buffer for example, Dulbecco's phosphate -buffered saline; DPBS
  • the advantage of this method is that (1) it uses a non-enzymatic solution - thus, there is no need for post-detachment washing or centrifugation to eliminate enzyme, and (2) it does not require scraping - the cells treated with Versene® EDTA can be washed off the surface.
  • the hESCs treated with Versene® EDTA and detached without scraping have higher post-detachment viability and re-attach to the new culturing surface much faster (minutes vs. hours) when passaged.
  • the Versene® EDTA passaging/harvesting method is not ideal. Versene® EDTA seems to breaks down cell-cell association faster than it breaks cell-surface bonding. If hESC culture is over-treated with Versene® EDTA (>9 minutes), a greater percentage of cells come off the surface as single cells rather than clusters or clumps. In order to avoid getting too many single cells, the treatment time normally is controlled between seven to nine minutes. After the removal of Versene® EDTA, in six-well plate or T-flask culture format, fluidic shear force generated by hosing with culture medium via manual pipetting is needed to dislodge the cells off the surface.
  • hESC culture in multilayer vessels cannot be manually sheared with culture medium as pipettes cannot be introduced inside the vessels. Instead, in this culture format, after Versene® EDTA is replaced with culture medium, vigorous tapping is applied to dislodge the cells. The mechanical force (tapping) has to follow the replacement of Versene® EDTA with culture medium immediately because Versene® EDTA treated hESCs quickly re-attach to the surface once they come in contact with culture medium. In fact, with the current state-of-art, it is only possible to harvest 40-70% of the entire culture in multilayer cell factories - dramatically impacting the yield of these very expensive cells.
  • the present invention provides a non-enzymatic reagent formulation and a method of harvesting and subsequently passaging pluripotent stem cells as clusters with high yield and high post-detachment cell viability.
  • a formulation for harvesting and subsequent passaging of human pluripotent stem cells without scraping and without substantial loss of viability.
  • the formulation includes, for example, sodium citrate, a salt, and a phosphate-buffered saline solution, at an osmolality of about 250-1050 mOsmol/Liter.
  • the osmolarity is 311-1014 mOsmol/Liter.
  • the formulation is used to harvest and passage embryonic stem cells and induced pluripotent stem cells.
  • the concentration of citrate is, for example, about 0.15 to 150 mMol/Liter.
  • the salt is, for example, NaCl, KCl, Na2HP03, NaH2P03, K2HP03, or KH2P03.
  • the concentration is, for example, about 1.00-1400 mMol/Liter
  • the concentration is about 1.35-1350 mMol/Liter.
  • the osmolarity of the formulation is, for example, about 400-700 mOsmol/Liter. In another aspect of the embodiment, the osmolarity of the formulation is, for example, about 418-570 mOsmol/Liter.
  • the formulation is pH buffered with, for example, bicarbonate, phosphate, ethanolamine, triethanolamine, or trometamol.
  • the pH of the formulation is, for example, about 7-8, In an alternative embodiment the pH is about 7.2-7.8.
  • the phosphate-buffered saline solution is, for example, Ca2+/Mg2+-free Dulbecco's phosphate buffered saline (DPBS).
  • treatment of the cells with the formulation results in the harvest of, for example, at least 90% of the cells from the surface of the culture vessel and cell viability of, for example, at least 90%.
  • a method for producing a formulation for harvesting and subsequent passaging of human pluripotent stem cells without scraping and substantial loss of viability.
  • a method for harvesting and subsequent passaging of hPSCs comprising incubating the hPSCs in one of the formulations described in the preceding paragraphs and in cell culture vessels for a period of time to allow the hPSCs to detach from the cell culture vessels in clusters with high yield and high post-detachment cell viability of, for example, about 85-100%.
  • the cell culture plates or vessels are, for example, petri dishes, multi-well cell culture plates, stacked cell culture apparatus, multilayer cell culture factories, and similar vessels known in the art to be capable of supporting the culture of hPSCs.
  • the cells are treated with the formulation for about 2-20 minutes.
  • the treatment time is 5-15min for hPSCs cultured in mTeSRI®. In another embodiment, the treatment time is 8- 20min for hPSCs cultured in StemPro®. (StemPro® and mTeSRI® are examples of commercially available defined media for hPSC culture.)
  • the cells are harvested in clusters with sizes ranging, for example, from about 10-1000 ⁇ . More particularly, the cluster size is, for example, about 40-500 ⁇ .
  • the method results in the harvest of, for example, at least 90% of the cells from the surface of the culture vessel and cell viability of at least 90%.
  • additional passaging and harvesting formulations including formulations containing EDTA and EGTA, other Ca2+ chelators besides sodium citrate, or combinations of various Ca2+ chelators. Two factors are identified related to cell detachment, the Ca2+ chelator concentration and osmolarity.
  • a method of developing a cell-detaching solution for hPSCs wherein the size distribution of the detached clusters and the percentage of the culture detached at given treatment time can be controlled with the osmolarity and Ca2+ chelator concentration.
  • the disclosed formulation includes sodium citrate, which disrupts the cell-surface bond and cell-cell association by chelating/sequestering Ca2+, the divalent cation required for cell-surface and cell-cell binding.
  • This sodium citrate -based formulation and process is specially designed and developed to address the unique challenges in routine or scale up hPSC culture and manufacturing processes.
  • hPSCs are normally passaged as multi-cellular clusters/aggregates, and passaging hPSCs as single-cells is to be avoided due to low cloning efficiency of hPSCs and the high risk of karyotypic abnormality.
  • This formulation and method is additionally optimized for harvesting and passaging hPSCs in reference to the key quality parameters of hPSCs, for example, viability, yield, post-detachment cluster size, passageability, and maintaining a pluripotent phenotype.
  • This formulation and process can be used in any hPSC lab as routine lab practice to expand hPSC culture with reduced labor intensity and process time.
  • this formulation and process does not require mechanical scraping to get the cells off the surface and the cell harvest does not need to be washed and centrifuged to remove the agents used to detach the culture.
  • This formulation and process especially benefit large-scale hPSC production when the cells are growing in multilayer cell culture vessels where scraping cannot be applied. Using this formulation and method, more than 90% of hPSCs grown in multilayer cell culture vessels can be harvested with more than 90% viability.
  • Non-enzymatic cell detachment solutions at various concentrations were screened with one objective being to improve the yield of hESCs harvested from multilayer culture vessels while retaining the simplicity of the Versene® EDTA harvesting/passaging method.
  • This screening included, for example, Versene® EDTA solutions at 0.1, 0.55, 1, 3, and 10 mM, Versene® based ethylene glycol tetraacetic acid (EGTA) solutions at 0.1, 0.55, 1, 3, and 10 mM, and 1 X sodium citrate solution (10X solution: 0.15 M sodium citrate, 1.35 M potassium chloride (KCI), diluted to 1 X in Ca2+/Mg2+-free DPBS).
  • EGTA ethylene glycol tetraacetic acid
  • KCI potassium chloride
  • EDTA, EGTA and sodium citrate are Ca2+ chelators and have been used historically for detaching adherent cells in culture.
  • Versene® EDTA has been used routinely for harvesting/passaging hESCs in some labs; both EDTA and EGTA (in combination with trypsin) were used to passage hESCs in a study published by Thomson et al. at Roslin Institute in Scotland in 2008 ( Thomson et al. (2008), "Human Embryonic Stem Cells Passaged Using Enzymatic Methods Retain a Normal Karyotype and Express CD30", Cloning and Stem Cells, 10 (1), 1-17 .).
  • hESCs cultured in Murine Embryonic Fibroblast-conditioned medium were treated with solutions including those described above as follows: (1) remove culture medium; (2) wash once with Ca2+/Mg2+ free buffer (for example, DPBS); (3) incubate in solution at room temperature; (4) remove solution; and (5) soak the colonies in MEF-CM for 0.5-1 minute to let them have a chance to stick back onto the surface. The colonies were then hosed with culture medium to see if they can be detached from the surface.
  • incubation step # 3 above it is noted that 4-9 minutes is the norm for Versene® EDTA.
  • step # 5 this step was executed in the initial detachment- solution screening study to see whether the cells re-stick onto the surface in culture medium.
  • the cells are hosed off the surface right after removing the cell-detachment solution.
  • the culture medium is poured into the vessel first, the medium is leveled in all layers, and then the vessel is tapped to dislodge the cells in the culture medium.
  • the pouring and leveling take 0.5-1 minute in a ten-layer vessel. In the cGMP setting, additional time may be required.
  • FIGURE 2 As illustrated by FIGURE 2 , after this procedure, 85% of the Versene® (0.55 mM EDTA)-treated culture remained on the surface ( FIGURE 2a ), while nearly all the cells came off the surface in 1 X sodium citrate (sodium citrate formulation) treated culture ( FIGURE 2b ). The visual assessment of the percentage of the culture remaining on the surface after treatment is plotted in FIGURE 2c .
  • sodium citrate solution is surprisingly identified as a superior reagent compared to Versene® EDTA for harvesting hESC culture.
  • Sodium citrate formulation disrupts the cell-surface bond to a greater extent than Versene® EDTA and sodium citrate formulation treated hESCs do not re-stick to the surface in culture medium as fast and easily as Versene® EDTA or EGTA formulation- treated hESCs.
  • FIGURE 3 shows that hESCs passaged with sodium citrate formulation for six times in MEF-CM retain typical hESC morphology.
  • FIGURE 4 the karyotype of this culture was evaluated to be normal after five passages with sodium citrate formulation.
  • FIGURE 5 provides images of the cells remaining on the surface after harvest.
  • the cultures in FIGURE 5a and 5b were both harvested using Versene® EDTA, but were handled by different operators (with the strength applied to tapping of stacks to dislodge cells varying between the operators).
  • the percentage of the culture detached from each two-layer stack is plotted in FIGURE 5d .
  • the number of cells harvested from each vessel was counted and the viability of the harvest was calculated ( FIGURE 5e ).
  • the counts of the cells harvested with IX sodium citrate solution have much tighter distribution (2.64E8+5.1E7 with Versene® EDTA vs. 3.46E8+1.81E7 with sodium citrate, (see FIGURE 5e ), which contributes to a more robust and consistent manufacturing process.
  • the yield of sodium citrate formulation-harvested hESC culture was 31% higher than that of Versene® EDTA harvested culture. There was less operator-to-operator variance when sodium citrate formulation was used. This may be because sodium citrate formulation disrupts the cell-surface bond more than Versene® EDTA and much less tapping effort was needed to dislodge the cells.
  • the optimal range for the concentration of sodium citrate for hESC detachment was 0.1X-3X (see Table 2 for the concentrations of sodium citrate and KCI), or higher.
  • sodium citrate formulations at higher sodium citrate concentrations did not necessarily detach the cells faster or break the colonies up into single cells more than lower concentrations ( FIGURE 6b ).
  • sodium citrate at higher concentration (1X-3X and higher) tended to lift the hESC colonies up as whole sheets.
  • the size of the detached hESC clump was dependent on the dilution of sodium citrate formulation more than treatment time (1-11 minutes tested).
  • clump size of the detached hESC grown in mTeSRI® was overall smaller than that grown in MEF-CM.
  • the effect of cell-detachment formulation on the size of detached clusters (clumps) of hESCs was further investigated. As described below, the osmolarity of the formulation affects the size of the clusters. In certain embodiments, a clump size of 10-1000 ⁇ m is preferred. In alternative embodiments, a clump size of 40-500 ⁇ m is desired.
  • the operational time window for sodium citrate formulation treatment on hESCs was wider than Versene® EDTA treatment on hESCs, which is particularly beneficial in cGMP-compliant production when product quality consistency is regulated and deviation is to be minimized.
  • the operational time window is further described below.
  • citrate formulations containing a salt other than KC1 are contemplated.
  • Such salts include, for example, NaCl, Na2HPO3, NaH2PO3, K2HPO3, KH2PO3, and the like.
  • sodium citrate concentration has a preferred working range of 0.15-150 mM.
  • sodium citrate concentration has a working range of 1.0-50 mM.
  • sodium citrate concentration has a working range of 1.5-45 mM.
  • sodium citrate concentration for passaging cells grown in MEF-CM is a working range of 1.5-15 mM.
  • sodium citrate concentration for harvesting cells grown in culture medium such as mTeSRI® is a working range of 1.5-30 mM.
  • Excipients in the formulation include, but are not limited to, Ca2+/Mg2+-free DPBS and KCl (1.35-1350 mM).
  • the working osmolarity of this formulation is from 31.1-2050 mOsmol/L. In one embodiment, the working range is 290-1015 mOsmol/L. In another embodiment, the working range is 299-781 mOsmol/L. In yet another embodiment, the range is 548-781 mOsmol/L.
  • sodium citrate solution of 10X contains 1350 mM KCl and 150 mM sodium citrate in water. A series of dilutions was made by diluting the 10X solution in DPBS (without Ca2+ or Mg2+).
  • Table 2 Formulations of Sodium Citrate Solution Dilution Factor 0.1x 0.3x 0.7x 1.0x 1.3x 2.0x 3.0x 10x KCl 13.5mM 40.5mM 94.5mM 135mM 175.5mM 270mM 405mM 1350mM Sodium Citrate 1.5mM 4.5mM 10.5mM 15mM 19.5mM 30mM 45mM 150mM
  • Osmolality of the screened cell detachment solutions was measured (Table 3).
  • Sodium citrate solution in general has a higher osmolality than all the Versene®-based EDTA and EGTA solutions (solutions screened in FIGURE 2 ). This may be due to the high concentration of KCl and other excipients in the solutions.
  • High osmolarity of the sodium citrate formulation is identified herein as one attribute that contributes to the unique cell detachment behavior of the solution, which is further discussed hereinafter.
  • Table 3 illustrates osmolarity of various solutions. Table 3.
  • hESC grown in MEF-CM
  • hESCs processed with kSep® remain as clusters and plate well with 10% drop in plating efficiency, compared with pre-kSep® fresh harvest ( FIGURE 9 ).
  • the process of expanding and passaging hESCs grown in mTeSR1® from T-flasks into multilayer cell factories is optimized with a novel non-enzymatic passaging method, followed by downstream processing with continuous counter-flow centrifugation technology.
  • hESC culture in MEF-CM is expanded, with Versene® EDTA passaging, from six-well plate into T-flasks, and further into multilayer cell culture vessels.
  • the final harvest from multilayer cell factories was characterized with flow cytometry and more than 90% of these cells expressed pluripotency markers including OCT4, SSEA4, Tra-1-60, and Tra-1-81.
  • the final cell harvest was concentrated more than 14 times after automated downstream processing, with only 2% drop in cell viability.
  • FIGURE 10 and 11 demonstrate the effect of the dilution of sodium citrate formulation on the trend of culture harvest increase with the increase of treatment time.
  • the study illustrated in FIGURE 10 was conducted on hESC culture grown in MEF-CM, and the study illustrated in FIGURE 11 was conducted on hESC culture grown in mTeSR1®.
  • the cells can be harvested from a variety of surfaces including but not limited to Matrigel-coated surface, gelatin-coated and MEF-plated surface, and/or chemically defined surfaces.
  • surfaces include, for example, synthetic plastic or peptide-bound surfaces (Corning Synthemax and Nunclon (Nunc) Vita surfaces), surfaces coated with defined recombinant or native proteins (e.g. vitronectin, fibronectin, laminin and collagen).
  • MEF-seeded surface MEF-seeded surface
  • Matrigel-coated surface Matrigel is an extracellular matrix derived from mouse tumor.
  • Synthemax and Nunc Vita are defined surfaces available on the market and marketed as a surface that can be used for hPSC culture.
  • Sodium citrate formulations can be used to harvest hPSCs grown in various types of culture medium, including, but not limited to, regular culture medium used for culturing hPSCs on MEF- seeded surfaces, MEF-CM, mTeSRI® and StemPro®.
  • the 1 X sodium citrate formulation described above is 15 mM sodium citrate, 135 mM KCI, pH 7.31, and 499 mOsmol/L in DPBS. Further studies were performed to determine the effects of osmolarity, KCl on the ability of the sodium citrate formulation to promote cell detachment.
  • FIGURE 12 shows the percentage of detachment of hESC in cultures treated with various solutions of sodium citrate. The results indicate that the osmolarity of the solution and the concentration of sodium citrate are important factors for achieving optimal cell detachment.
  • FIGURE 14B microscopic images revealed substantially larger clusters of harvested cells from cultures treated with 781-1016 versus 169-548 mOsmol/L solutions of sodium citrate. The images of the detached clusters were analyzed and the sizes of the clusters were quantified. The distribution of the cluster sizes is shown in FIGURE 14C .
  • FIGURE 15B microscopic images revealed substantially larger clusters of harvested cells from cultures treated with 30-75 mM sodium citrate solutions of sodium citrate.
  • FIGURE 15C There was a corresponding increase in the measured sizes of clusters obtained from cultures treated with 30-75 mM sodium citrate solutions versus 1.5-15 mM sodium citrate solutions (mostly 250-900 equidiameter size clusters versus mostly 40-150 equidiameter size clusters, respectively) ( FIGURE 15C ).
  • the Ca2+ chelation capacity of citrate, EDTA, and EGTA are 1: 1.5, 1: 1, and 1: 1, respectively, based on stoichiometry.
  • EDTA also binds Mg ⁇ 2+> and other metals whereas EGTA is more specific for binding of Ca2+.
  • Cell detachment achieved by a solution of sodium citrate (15 mM sodium citrate at 400 mOsmol/L (achieved by addition of KC1) and a pH of 7.8) was compared to that achieved by 22.5 mM and 45 mM solutions of EDTA and EGTA at 400 mOsmol/L and a pH of 7.8.
  • FIGURE 16A EDTA and EGTA at concentrations higher than 22.5 mM detached hESC colonies as effectively as sodium citrate.
  • the measured sizes of redistributed clusters obtained from cultures treated with the various solutions were similar (mostly 40-200 equidiameter size clusters) ( FIGURE 16B ).
  • FIGURE 18A shows the effects of Versene EDTA and various sodium citrate solutions on hESC percent detachment at each treatment time point.
  • FIGURE 18B shows the effects of Versene® EDTA and various sodium citrate solutions on cluster size distribution at each treatment time point.
  • FIGURE 19 shows the effects of Versene® EDTA and various sodium citrate solutions on the plating efficiency of detached cells at each treatment time points of 5, 10 and 20 minutes.
  • an operational window similar to that depicted in FIGURE 20A can be established and appropriate parameters can be selected to achieve a desired percentage of cell detachment and cluster size (equi-diameter).
  • the criteria used to determine the operational window is (1) the percentage of culture detached is over 70% and (2) the percentage of culture detached as 40-500um clusters is over 90%.
  • the durations of the operational windows of 22.5mM EGTA and Versene® EDTA formulations are similar to that of citrate formulation Dis2#3, and are much wider than that of Versene® EDTA.
  • the starting time points of the operational windows of 22.5mM EGTA and EDTA formulations are the same, and are earlier than that of citrate formulation Dis2#3.
  • sodium citrate formulations outperform EDTA- containing and EGTA-containing formulations in passaging hPSCs. Furthermore, another advantage of sodium citrate over EDTA and EGTA is that leftover sodium citrate should have less deleterious effects on cell cultures than EDTA or EGTA. (Sodium citrate is sometimes used as a normal component in various culture media of various cell types.) For example, as seen in EXAMPLE 6, sodium citrate formulation solutions do not necessarily need to be removed after treating the cells.
  • additional passaging and harvesting formulations are contemplated herein including formulations containing EDTA and EGTA, other Ca2+ chelators besides sodium citrate, or combinations of various Ca2+ chelators.
  • concentration is too low, the bond between the cells and the matrix is not effectively disrupted and the formulation cannot effectively detach the cells.
  • concentration is too high, the bond between the cells is overly disrupted, the size of the detached clusters decrease, and the detached clusters can be fragile and easily disintegrated when further handled.
  • the shrinking of the cells or the colonies caused by high osmorality can result in cell detachment, especially at the edge of the colonies.
  • the cells could also be damaged if the osmolarity is too high.
  • the osmolarity is too high, the cells are not effectively detached from the surface.
  • EXAMPLE 4 provides studies toward further optimization. Through use of the experimental design disclosed herein, other cell-detaching/passaging formulations containing various Ca2+ chelators can be developed and are considered within the scope of the present invention.
  • Loss of the cells can occur during treatment with cell-detaching solutions. This is because the culture is soaked in the solution during treatment, and the solution is removed prior to detaching the culture with cell culture medium. In order to eliminate this loss, studies were performed to find out whether the solution can be added and removed right away at the beginning of the treatment, and whether the culture can be detached with only the residue of the solution on the culture surface ("dry passaging").
  • the proposed new procedure is depicted in FIGURE 22 . As also indicated in FIGURE 22 , two treatment time points (5, 10 min), two incubation conditions (Room Temperature, 37°C incubator) were compared, and both Versene® EDTA and citrate formulation Dis2#3 were tested for this dry passaging procedure.
  • FIGURE 24 depicts the methodology used in "leave- in passaging”. Basically, after soaking the culture with the cell-detaching solution for a certain treatment time, the solution is not removed from the culture; instead, it is left in the culture and neutralized with culture medium. The cells are then dislodged from the surface by a swirling motion, and seeded onto a fresh surface afterwards. The cell-detaching solution is left in the final cell harvest and is later transferred into the next passage of the culture when the cell harvest is seeded onto fresh surface.
  • citrate is sometimes used as a normal component of some cell culture medium, the remaining citrate should not have deleterious effects on cell cultures.
  • Such a protocol provides increased ease-of-use and scalability of the sodium citrate formulations for cell detachment.
  • the solutions are not required to be removed from the culture vessel and tapping of the vessel is not used to dislodge the cells.
  • the protocol could be applied in small scale bench-top culture plate culture format; it could also be applied in, for example, harvesting cells grown in 10-layer, or even 40-layer cell culture vessels that have to be handled by robots (e.g. Automatic Cell Factory Manipulator).
  • FIGURE 24 The study described in FIGURE 24 was conducted on hESC culture grown in StemPro® medium. The culture was treated with two sodium citrate formulations, solution #3 and #13 described in EXAMPLE 4, at the volume of 0.5mL and 0.75mL.
  • FIGURE 25 shows the post-detachment viable cell number and percent viability achieved using leave-in passaging with the two sodium citrate formulations.
  • FIGURE 26 shows the post-detachment cluster size distribution and plating efficiency achieved using leave-in passaging with the two sodium citrate formulations.
  • citrate formulations described above were initially identified and optimized for applications in harvesting and passaging hESCs grown in large-scale in closed culture vessels.
  • culture vessels were agitated by tapping, swirling or shaking in the experiments to simulate the cell detachment process in closed culture vessels.
  • open culture platforms e.g. six-well plates or T-flasks, are often used, and cells are accessible to be dislodged by hosing with a stream of culture medium, as described in Table 1 under "EDTA method".
  • citrate solution #3 and #13 were tested in the small-scale bench- top applications, and compared their performance with Versene® EDTA.
  • the culture was grown in mTeSR1® medium and was treated for 5-8 minutes with the formulations followed by hosing (note that for large scale application, longer treatment, around 10 min, may needed).
  • Treatment of hESC cultures with either solution for 5 minutes followed by hosing with a pipette works well and achieves 95% harvesting and approximately 70-85% replating efficiency.
  • the replating efficiency of citrate solution #13 was about 15% higher than that of Versene® EDTA.
  • hESCs cultures in StemPro® and mTeSR1® were initiated and maintained for over 50 passages (about 8 months) by continuously passaging with citrate formulations.
  • Sodium citrate solutions Dis2#3, #3 and #13 were evaluated and Versene® EDTA was included in this study as a comparison to the citrate formulations.
  • FIGURE 29 hESCs passaged with citrate solution #13 for 31 passages in StemPro® medium, and 34 passages in mTeSR1®, respectively, stained positive for hESC markers, including OCT4, Sox2, Nanog, SSEA4, TRA-1-60 and TRA-1-81). The marker expressions were further quantified with flow cytometry.
  • Pluripotency of these hESCs was further evaluated for differentiation capability.
  • hESCs passaged with citrate solution for 40 passages in StemPro® and 35 passages in mTeSR1®, respectively were capable of generating embryoid bodies (EBs) and differentiating into all three germ layers, endoderm, mesoderm and ectoderm.
  • the differentiation capability of the long-term hESC culture in StemPro® was further confirmed by the generation of teratomas of these cells in immunodeficient SCID mice ( FIGURE 31 ).
  • the results from EB differentiation and teratoma generation proved that hESCs passaged with citrate solution for numerous passages and prolonged time are still pluripotent and retain the capability of differentiating into all kinds of somatic cells.
  • G-banding karyotype of the long term hESC culture passaged with citrate solution was examined for abnormality.
  • Three samples of hESCs cultured in StemPro® medium were karyotyped normal after passaged for 10 and 25 passages with the citrate solution #13; in contrast, whereas three samples of hESCs are normal after 10 passages with Versene® EDTA, one out of three samples are abnormal and one sample had emerging abnormality after 25 passages with Versene® EDTA.
  • HESCs cultured in mTeSR1® medium were also karyotyped normal after passaged for 29 times with the citrate solution #13.
  • the split ratio used to passage the long term hESC cultures was 1:15 to 1:40 (i.e. for example, cells harvested from one well could be split into 15 to 40 fresh wells; in the context of hPSC cultivation, 1:40 is considered higher split ratio than 1:15), in both mTeSRI® and StemPro®.
  • the cultures typically reached confluence on day 4 or day 5 after split.
  • the split ratio is typically 1:2 to 1:6, and the culture typically reaches confluence between day 5 and day 7.
  • the passaging method disclosed herein results in much faster expansion of the culture.
  • sodium citrate formulations were tested on other hESC lines (in addition to H9) and human iPSCs.
  • Sodium citrate solution #13 was used to passage H7 and H14 hESCs grown in mTeSR1®. Unlike the H9 cultures used in the other studies, the starting cultures of H7 and H14 had many stroma or differentiated cells. The citrate solution selectively detached and passaged mostly the undifferentiated hESCs, and left the stroma and differentiated cells on the surface. Following three consecutive passages with the solution, the morphology of both the H7 and H14 cultures was greatly improved with no obvious stroma or differentiated cells observed. It follows that the sodium citrate formulations can be used to detach and passage various hESC lines.
  • Citrate solution #13 can be used to detach and passage iPSCs. Even when the cells were transferred from MEF feeders onto Matrigel, the attachment was good when the detached cells were re-plated, and the majority of the MEF feeders was not detached and hence not subsequently transferred onto the fresh Matrigel surface. In this experiment, the human iPSC culture was also gradually adapted into defined medium (StemPro®) in five days at passage 1. It therefore follows that the citrate formulations can be applied onto various human iPSC lines for passaging and maintaining the established culture.
  • the formulation can further be used to transfer the hPSC cells, including hESCs and human iPSCs, from MEF feeders onto feeder- free surfaces, like Matrigel, with reduced time to clear up the remaining MEFs (compared with mechanical passaging methods involving scraping).
  • MEF feeders including hESCs and human iPSCs
  • feeder- free surfaces like Matrigel
  • the images of the Day 1 re-plated cultures also indicated that the hESC clusters harvested with the citrate solution were not fully disintegrated into single cells or smaller clusters by KSep processing.
  • sodium citrate solution # 13 is capable of generating robust clusters that survive kSep®, automated vialing and cryopreservation with Controlled-Rate Freezer, retain high viability and plating efficiency, and present normal karyotype post-scaling up.

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Claims (17)

  1. Procédé pour la récolte et le passage subséquent de cellules souches pluripotentes humaines sans grattage et sans perte sensible de viabilité, comprenant :
    l'incubation desdites cellules souches pluripotentes humaines dans une formulation, ladite formulation comprenant :
    du citrate de sodium, et un sel, lequel sel est choisi dans le groupe constitué par NaCl, KCl, Na2HPO3, NaH2PO3, K2HPO3, KH2PO3 et NaHCO3,
    dans lequel ladite formulation a une osmolarité de 150 à 1500 mOsmol/litre.
  2. Procédé selon la revendication 1, dans lequel ledit citrate de sodium est à une concentration de 0,15 à 150 mmol/litre.
  3. Procédé selon la revendication 1 ou 2, dans lequel le citrate de sodium est à une concentration de 1 à 30 mmol/litre, de préférence à une concentration de 1 à 15 mmol/litre.
  4. Procédé selon la revendication 1, dans lequel l'osmolarité est de 500 à 700 mOsmol/litre, de préférence de 418 à 570 mOsmol/litre.
  5. Procédé selon la revendication 1, dans lequel les cellules souches pluripotentes humaines sont choisies dans le groupe constitué par les cellules souches embryonnaires et les cellules souches pluripotentes induites.
  6. Procédé selon la revendication 1, dans lequel le sel est KCl et dans lequel le KCl est à une concentration de 1,35 à 1350 mmol/litre, de préférence à une concentration de 203 à 316 mmol/litre.
  7. Procédé selon la revendication 1, dans lequel la formulation a un pH tamponné avec du bicarbonate, du phosphate, de l'éthanolamine, de la triéthanolamine, ou du trométamol.
  8. Procédé selon la revendication 1, dans lequel la formulation a un pH compris entre 7 et 8.
  9. Procédé selon la revendication 1, comprenant en outre une solution salée tamponnée au phosphate, qui est une solution salée tamponnée au phosphate de Dulbecco (DPBS) sans Ca2+/Mg2+.
  10. Procédé de production d'une formulation pour la récolte et le passage subséquent de cellules souches pluripotentes humaines, comprenant :
    la combinaison de citrate de sodium et d'au moins un sel avec un liquide de façon que l'osmolarité soit de 150 à 1500 mOsmol/litre et que la concentration de citrate de sodium soit de 0,15 à 150 mmol/litre, lequel sel est choisi dans le groupe constitué par NaCl, KCl, Na2HPO3, NaH2PO3, K2HPO3, KH2PO3 et NaHCO3.
  11. Procédé pour la collecte et le passage subséquent de cellules souches pluripotentes humaines selon la revendication 1, comprenant :
    l'incubation des cellules souches pluripotentes humaines dans une formulation comprenant :
    du citrate de sodium, et un sel, lequel sel est choisi dans le groupe constitué par NaCl, KCl, Na2HPO3, NaH2PO3, K2HPO3, KH2PO3 et NaHCO3,
    dans lequel ladite formulation a une osmolarité de 150 à 1500 mOsmol/litre, dans des cuves ou des plaques de culture cellulaire pendant 2 à 20 minutes, dans lequel lesdites cellules souches pluripotentes humaines se détachent des cuves ou plaques de culture cellulaire en grappes ayant une viabilité cellulaire comprise entre 85 et 100 %, et dans lequel la taille de grappe moyenne est de 10 à 1000 µm.
  12. Procédé selon la revendication 11, dans lequel les cuves ou plaques de culture cellulaire sont choisies dans le groupe constitué par les boîtes de Pétri, les plaques de culture cellulaire à puits multiples, les dispositifs de culture cellulaire empilés, et les usines de culture cellulaire.
  13. Procédé selon la revendication 11, dans lequel la taille de grappe moyenne est de 40 à 500 µm.
  14. Procédé selon la revendication 11, comprenant en outre :
    un traitement en aval des grappes, lequel traitement en aval est choisi dans le groupe constitué par une technologie de centrifugation à contre-courant en continu, une formulation, une mise en flacon automatisée, et une cryoconservation.
  15. Procédé de production d'une formulation pour la récolte et le passage subséquent de cellules souches pluripotentes humaines selon la revendication 1, comprenant :
    le passage des cellules souches pluripotentes humaines avec la formulation comprenant du citrate de sodium, et un sel, lequel sel est choisi dans le groupe constitué par NaCl, KCl, Na2HPO3, NaH2PO3, K2HPO3, KH2PO3 et NaHCO3,
    dans lequel ladite formulation a une osmolarité de 150 à 1500 mOsmol/litre, avec un rapport de division de 1/10 à 1/60, et dans lequel la culture atteint la confluence en l'espace de sept jours après la division.
  16. Procédé pour la récolte et le passage subséquent de cellules souches pluripotentes humaines selon la revendication 1, comprenant :
    le placage de cellules souches pluripotentes humaines dans un milieu,
    l'aspiration du milieu usé,
    le lavage avec de la DPBS,
    l'addition de la formulation comprenant du citrate de sodium, et un sel, lequel sel est choisi dans le groupe constitué par NaCl, KCl, Na2HPO3, NaH2PO3, K2HPO3, KH2PO3 et NaHCO3, laquelle formulation a une osmolarité de 150 à 1500 mOsmol/litre,
    le retrait de la formulation, l'incubation,
    l'addition de milieu de culture, et
    la collecte des grappes détachées.
  17. Procédé selon la revendication 11, dans lequel la formulation n'est pas retirée.
EP12723066.2A 2011-05-17 2012-05-17 Formulation et procédé de passage et de collecte de cellules souches pluripotentes humaines Active EP2718424B1 (fr)

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CA3050259A1 (fr) * 2017-01-17 2018-07-26 Lonza Walkersville, Inc. Formulation de repiquage et de recolte pour cellules souches pluripotentes humaines isolees
JP2018201403A (ja) * 2017-06-02 2018-12-27 日本ゼオン株式会社 幹細胞の培養方法
EP4073234A1 (fr) * 2019-12-11 2022-10-19 Repairon GmbH Multiplication de cellules souches cultivées en suspension dans un bioréacteur
WO2023070016A1 (fr) * 2021-10-20 2023-04-27 Exir Llc Compositions et procédés d'utilisation d'ensembles génomiques et de lignées de cellules souches pluripotentes induites individualisés de primates non humains pour une évaluation préclinique
WO2023145797A1 (fr) * 2022-01-31 2023-08-03 キヤノン株式会社 Procédé de détachement de cellules, système de détachement de cellules et dispositif de traitement de l'information

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JP6140149B2 (ja) 2017-05-31
AU2016238891B2 (en) 2019-05-09
WO2012158899A1 (fr) 2012-11-22
ES2703751T3 (es) 2019-03-12
IL229462A0 (en) 2014-01-30
SG194967A1 (en) 2013-12-30
CA2836578C (fr) 2019-09-10
AU2012255724A1 (en) 2013-11-28
IL229462B (en) 2019-08-29
US20140212966A1 (en) 2014-07-31
AU2012255724B2 (en) 2016-07-07
AU2016238891A1 (en) 2016-11-10
US9885019B2 (en) 2018-02-06
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EP2718424A1 (fr) 2014-04-16
US20180171300A1 (en) 2018-06-21

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